Fluorescent dyes form the building blocks of many reagents that are used in a myriad of bioanalytical applications such as nucleic acid detection and sequencing, flow cytometry for cellular characterization, fluorescence microscopy, enzymatic assays and increasingly in the field of optical imaging as probes to detect disease tissue and organ in vivo. There are a large number of fluorescent dyes available for use in microscopy, immunohistology, and other high technology research. These dyes have extended conjugated carbon chains embedded in their chemical structures. The molecules are able to absorb light energy and emit light of a different colour. The emission wavelengths of organic dyes are usually fine-tuned to emit light of longer wavelength by incorporation of an electron sink within the molecule that allows for delocalization of π electrons along the unsaturated chain. Even though NIR dyes have been developed for many years for use in high technology fields, just a handful of them have found use for in vivo applications.
Optical imaging, in particular fluorescence offers several advantages that make it a powerful molecular imaging approach, both in the research and clinical settings. Specifically, optical imaging, besides being fast, safe, cost effective and highly sensitive, can be tailored for diagnostic as well as therapeutic outcomes. The organic dyes typically used as imaging reporters are amenable to design modifications through various linker chemistries to incorporate one or more targeting motifs. Fluorescence imaging is translational from the preclinical stage in small animals to human subjects as the same agent can be used without modifying the biological target. While bioluminescence has the sensitivity it is not translatable from preclinical small animal imaging to humans. Also the luciferin/luciferase based system cannot be multiplexed to interrogate multi mode based mechanism of binding to cell surface or of receptors in tumours for example in oncology applications. Fluorescence based methods are thus a natural choice to bridge the translation of imaging reagents used concurrently with the established PET, SPECT MRI and X-ray methods as the optical reporter dyes are amenable via multiple linker chemistry to carry similar or different recognition motifs. Molecular imaging involves the use of a “molecular” probe or agent that selectively targets a particular cellular receptor, nucleic acid sequences of a gene, amino acid sequences and post translational motifs within a protein, epigenetic modifications, cellular function and pathways, with the absence, presence or level of the specific target being indicative of a particular disease state.
The development of NIR fluorescent dyes has played a critical role in the optical imaging field, allowing it to become an increasingly important contributor to imaging science. In contrast to the classical application of fluorescent dyes in other technologies; the design of fluorescent dyes for in vivo applications needs to incorporate several important criteria including (1) water solubility, (2) structural and chemical stability, (3) NIR fluorescence, (4) high quantum yield and last but, not least (5) a functional group for bioconjugation.
Among the fluorescent dyes available for optical imaging the cyanine family of dyes have been the preferred class as they provide the wavelength range for in vivo fluorescence excitation and emission not compromised by the optical properties of the tissue of interest. Hemoglobin has a strong absorption at wavelengths lower than 600 nm and significant background fluorescence from endogenous biomolecules can be detected up to 650 nm. The heptamethine cyanine dyes, which absorb and emit beyond 750 nm, are classified as near infrared (NIR) dyes and are preferred labels for in vivo imaging as near infrared light can overcome the biological optical interference limitations by penetrating more deeply into tissue, because light scattering decreases with increasing wavelength.
Cyanine dyes are characterized as possessing two heterocyclic moieties, acting as both electron donors and acceptors, and are joined by a single or odd of number of methine groups in which (n+1) 2 electrons are distributed over n atoms producing a delocalized cation across the methine chain. This unique characteristic gives cyanine dyes a wider range of absorption than any other known class of dyes. A great number of synthetic cyanines are known to absorb between the visible and infrared regions of the electromagnetic spectrum. In addition, cyanines exhibit narrow absorption bands and high extinction coefficients. Due to these properties, cyanine dyes have been extensively employed in various applications such as photographic processes, laser printing, nonlinear optical materials, and more recently fluorescent probes for biomolecular labelling. For example, U.S. Pat. No. 5,571,388 discloses exemplary methods of identifying strands of DNA by means of cyanine dyes. More recently, they have been used for optical imaging of dye-labelled biomolecules, either in vivo or in vitro (U.S. Pat. No. 7,597,878, others). Cyanine dyes are the preferred labels in biological applications because, among other reasons, many of these dyes fluoresce in the near-infrared (NIR) region of the spectrum (600-1000 nm).
Development of polymethine cyanine dyes that absorb longer wavelengths for in vivo imaging applications, have focused on polyenes since each double bond enhancement in this region increases the bathochromic shift by ˜100 nm. This feature shows the advantage of cyanines compared to other dyes where tuning is contingent upon the expansion of the aromatic rings. Several lines of research have demonstrated that addition of an aromatic 6-membered ring would shift the absorbance by approximately 20 nm. The major drawback to this approach is the increased hydrophobicity of the resulting compound.
Advantages of cyanine dyes include, for example: 1) strong absorption cross sections and ability to fluoresce after excitation; 2) they do not rapidly bleach under a fluorescence microscope or plate reader excitation sources; 3) the derivatives are amenable as effective coupling reagents without loss of photochemical properties; 4) many structures and synthetic procedures have been developed over the last sixty years, and the class of dyes are versatile reagents; 5) cyanine dyes are relatively small (a typical molecular weight is about 1,000 daltons), so they do not cause appreciable steric interference in a way that might reduce the ability of a labelled biomolecule to reach its binding site or carry out its function and 6) when appropriately derivatized are not pH sensitive.
However, many of the known cyanine dyes have a number of disadvantages, such as chemical instability in the presence of certain reagents that are commonly found in bioassays. Such reagents include ammonium hydroxide, dithiothreitol (DTT), primary and secondary amines, and ammonium persulfate (APS). Further, some cyanine dyes lack the thermal stability and photostability that is necessary for biological applications such as DNA sequencing and genotyping. Besides photostability, which arises due to the cis trans summarization or disruption of the extended conjugation, aqueous solubility and charge modification are sometimes needed to derive superior biomedical applications.
For these reasons, there is still a need for stable cyanine dyes for use in labelling biomolecules as well as in vivo imaging for the diagnosis and prognosis of diseases such as cancer, infectious disease imaging and metabolic activity. Such compositions and methods would aid in the analysis of responses to various therapies.
U.S. Pat. No. 5,217,846 photopolymerizable compositions containing initiator systems that absorb in the longer wavelength region of the visible spectrum. The photopolymerizable composition comprises at least one ethylenically unsaturated monomer capable of free radical initiated addition polymerization and an initiator system activatable by actinic radiation, wherein said initiator system comprises a hexaarylbisimidazole, a coinitiator, and a sensitizer.
TOLMACHEV et al report in a “KHIMIYA GETEROTSIKLICHESKIKH SOEDINENII”—“CHEMISTRY OF HETEROCYCLIC COMPOUNDS”, LATVIJSKIJ INSTITUT ORGANICESKOGO SINTEZA, RIGA, LV, the syntheses of derivatives of glutaconaldehydedianil hydrochloride of general formula I, where α,α′-carbon atoms are included to dihydropyran, dihydrothiopyran, and N-methyltetrahydropyridine cycle.

It was shown that synthesized dianils give the corresponding cyanine dyes on heating with 2-methyl-3-ethylbenzothiazolium or 2-methyl-3-ethylnaphtothiazolium tozylates under basic conditions (sodium acetate or triethylamine in dry ethanol) of the general formula below:
